The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

Hénault, Camille M.; Juranka, Peter F.; Baenziger, John E.

doi:10.1074/jbc.m115.676833

Cited by 26 publications

(49 citation statements)

References 71 publications

(72 reference statements)

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“…The structural changes observed in EPR are consistent with the study in GLIC M4 that shows that mutational perturbations in the extracellular half of M4 have a greater effect on pH 50 in comparison to the intracellular end (Hénault et al, 2015). The proposed M4 conformational change is also consistent with the lipid-mediated M4 tilt observed in isolated peptides (Antollini et al, 2005).…”

Section: Discussionsupporting

confidence: 87%

“…Proline-induced local distortion of the helix and increases in flexibility are predicted to have global effects on protein conformational changes (Cordes et al, 2002). Consistent with this idea, mutational perturbation of the conserved proline side-chain leads to non-functional channels (Hénault et al, 2015), suggesting that the helix-bending at this position may play a role in channel gating. Above this region (towards the extracellular end), for the TMD-facing side of M4, a large change in lineshape (observed as a decrease in spectral broadening and an increase in amplitude; also see the changes in mobile and immobile spectral components marked by the dotted lines) is noted at several positions (Phe299, Val302, Phe303, Phe314, and Phe315) and modest changes are observed at others (Ala306, Asn307, and Leu310).…”

Section: Resultsmentioning

confidence: 77%

“…Single-cysteine mutations were made along the length of the segment (~31 positions) on a cysteine-less background template. Previous studies have also shown that M4 mutations are well-behaved, with the exception of Pro300, where no pH-activated currents were observed (Carswell et al, 2015; Hénault et al, 2015) Individual cys-mutants were purified, labeled with MTSL, and reconstituted into asolectin membranes for EPR studies. As with any study involving side-chain perturbations, attachment of spin-probes could lead to functional alterations of the channel.…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel

Basak

Schmandt

Gicheru

et al. 2017

eLife

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Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω−3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.DOI: http://dx.doi.org/10.7554/eLife.23886.001

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Section: Discussionsupporting

confidence: 87%

Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

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Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel

Basak

Schmandt

Gicheru

et al. 2017

eLife

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“…The final sensitive residue identified in GLIC, Pro-299, is located in the middle of the M4 helix ( Fig. 6 A ) and has recently been identified as functionally important in an alanine screen of the M4 helix ( 31 ). Although a homologous proline does not appear in the nAChRs or 5-HT 3 Rs, it is conserved in the GABA A Rs, GlyRs, and ELIC.…”

Section: Resultsmentioning

confidence: 99%

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Rienzo

Rocchi

Threatt

et al. 2016

Journal of Biological Chemistry

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Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

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“…1 ) and there have been a range of studies on these, with most of them being concerned with the residues in M4. Studies suggest that M4 interactions with M1 and M3 in some pLGICs are necessary to promote effective interactions between the M4 C-terminus and the Cys-loop, which are located at the interface of the ECD and the TMD, and are important in coupling agonist binding to channel opening [ 19 , 20 ]. It is also possible that there are more direct links, for example W418 in the nAChR M4 α-helix has recently been shown to interact directly with S226 on the adjacent M1 α-helix, stabilizing the open state [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

The roles of aromatic residues in the glycine receptor transmembrane domain

Tang

Lummis

2018

BMC Neurosci

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BackgroundCys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1–M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues.ResultsHere we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC50s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data.ConclusionThe data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function.Electronic supplementary materialThe online version of this article (10.1186/s12868-018-0454-8) contains supplementary material, which is available to authorized users.

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The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

Cited by 26 publications

References 71 publications

Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel

Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

The roles of aromatic residues in the glycine receptor transmembrane domain

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